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96 Well Plate Worm Lifespans
Lifespans in 96 well plates-'' C. elegans '' Protocol adapted from Greg M. Solis and Michael Petrascheck from The Scripps Research Institute. Day -7: Thursday (week 1): Inoculate 5 mL of TB containing 100 μg/ mL Ampicillin P/N: (Sigma-Aldrich, A0166-25G, dessicator in 4°C , #3 ) and 0.1 μg/ mL Amphotericin B P/N: (Sigma-Aldrich, A2942, dessicator in -20°C , #2 ) with a single OP50 colony and incubate over night at 37°C in a bacterial shaker. Day -6: Friday (week 1). Morning 8:30. Inoculate early to allow enough time for the culture to reach saturation 1. Dilute the overnight culture of OP50 1:2000 in 300 mL TB containing 50 μg/ mL Ampicillin. Incubate the culture in a bacterial shaker for 8-12hours at 37°C until saturation is reached. Do not allow the culture to grow longer than 14 hours. 2. Transfer the OP50 into a sterile, pre-weighted centrifugation tube. Pellet the OP50 by centrifugation for 10 min at 3500 rpm (2200 x g) in a table top centrifuge. 3. Discard the supernatant and re-suspend the OP50 pellet in sterile water and re-pellet by centrifugation. Repeat this wash twice. 4. After the second wash, carefully remove all the remaining water. No water should be left in the tube. Weigh the centrifugation tube containing the pellet and subtract the weight of the empty centrifugation tube in order to determine the weight of the pellet. 5. Thoroughly re-suspend the pellet in S-complete to a concentration of 100 mg/ mL. No clumps should be left. 6. The concentration of 100 mg/ mL OP50 should correspond to 2 x1010 bacteria/ mL. Use a photo spectrometer to determine the number of bacteria per mL if the relationship between the optical density and number of bacteria per mL is known. If necessary, adjust the concentration of the OP50 feeding solution to 2 x1010 bacteria/ mL. 7. Store the OP50 solution at 4°C until it is used for the worm culture. Afternoon 4:00 p.m.: Transfer the animals to a fresh plate 1. Take a 5-10 day old NGM plate on which the majority of the worm population consists of starved L1 larvae. 2. Sterilize a metal spatula by shortly heating it over a Bunsen burner. Let it cool. Use the cooled spatula to cut out agar chunks from the plate containing the starved worms. Transfer several of these chunks onto a fresh 10 cm NGM plate seeded with OP50. Incubate for approx. 65 hours at 20 °C until the majority of the worm population consists of gravid adults. The time it takes for the starved L1 animals to grow into gravid adults may vary from strain to strain. Day -3: Monday (week 2) 10:00 a.m.: Establish a synchronous population 1. Collect worms from the 10 cm plate by washing them off the plate with 5-10 mL sterile water. Transfer the worm/water solution into a 15 mL conical tube. 2. Wash the worms by centrifuging 2 min in a tabletop centrifuge at 1200 rpm (280 x g). Discard supernatant and add 10 mL water. Repeat. 3. Remove supernatant and add 5 mL of freshly prepared bleach/NaOH solution (2.5 mL household bleach, 1 mL 10N NaOH, 6.5 mL H2O). Incubate for 5 minutes at RT until the worms break open. Be sure to vortex gently every minute. Monitor the progress of the reaction under the dissecting microscope. 4. As soon as all the adults dissolve, add 5 mL of M9 buffer to neutralize the reaction. 5. Wash the eggs three times with 10 mL M9 buffer by centrifuging 2 min at 2500 rpm (1100 x g). 6. Wash the eggs once with 10 mL S-complete by centrifuging 2 min at 2500 rpm (1100 x g). 7. Remove the supernatant, add 10 mL S-complete LINK TO RECIPE FOR S-COMPLETE WITH P/N's and transfer the solution to a fresh 50 mL conical tube. Add 30 mL of S-complete to a final volume of 40 mL. Gently shake the tube overnight at room temperature on a nutator or similar device. Day -2: Tuesday (week 2), 12:00 noon: Seed the animals into plates 1. Under the dissecting scope, check whether the worms hatched during the night. Determine the concentration of worms in the S-complete solution by counting the number of worms in 10 μL drops using a dissecting scope. Count at least 10 drops for each sample. 2. Re-suspend the worms at a concentration of 80-100 worms/ mL in S-complete. Add Carbenicillin (plantMedia, 40310000-3, dessicator in 4°C, #3) (stock 100 mg/ mL) to a final concentration of 50 μg/ mL and Amphotericin B (SIGMA A2942, -20C dessicator in #2, stock 250 μg/ mL) to a final concentration of 0.1 μg/ mL. Shake on a nutator. If preparing larger quantities of worms, use a 600 mL nunclon flask with filter cap. 2b. Add the correct color for your plate according to the Worm plate color code 3. At 2:30 p.m. add the OP50 prepared in Part 1 to a final concentration of 6 mg/ mL (= 1.2 x109 bacteria/ mL). Return the OP50 to 4°C. 4. Transfer 120 μL of the worm/OP50 solution into each well of a 96 well plate. Use 96 well plates with a transparent bottom (Olympus #25-104, vis Genesee Scientific). Make sure to keep the worms in suspension while pipetting. 5. Seal the plate using a tape sealer to avoid contamination and evaporation. Shake the plate on a microtiter plate shaker for 2 min and incubate for 2 days at 20°C until the animals reach the L4 stage. Day 0: Thursday ' '(Week 2) Before noon: Sterilize animals by adding Fluorodeoxyuridine (FUDR) 15. To sterilize the animals add 30 μL of a 0.6 mM FUDR (Bioworld, 40690016-1, dessicator in 4°C, #3) stock solution to each well. This step brings the final volume in each well to 150 μL and reduces the final concentration of OP50 from 6mg/ mL to 5 mg/ mL (1x109 bacteria/ mL). Seal the plate using tape sealers and shake it for 2-3 min on a microtiter plate shaker. If the OP50 was added at 2:30 on day -2, it is important that the FUDR is added before noon. Return plates to the 20°C incubator Scoring of Lifespans 1. On day 0, at the beginning of the experiment count the total number of worms in each well. Censor wells that contain more than 18 animals from the analysis as animals in these wells will not have enough OP50 and will show effects of dietary restriction. 2. In order to increase the chance that the animals move, shake the 96 well plate on a microtiter plate shaker for 2 min. before counting. Day 1: Friday (week 2): Add drugs to culture 1. By 9:00 a.m. most of the animals should be gravid and contain several eggs each. Add the drugs whose effect on lifespan is to be tested at the desired concentration. If dissolving the drugs in DMSO the final concentrations of DMSO should not exceed 0.6%, as DMSO concentrations higher than 0.6% shortens C. elegans lifespan. After addition of the drug, seal the plates with tape sealer and shake it for 2-3 min on a microtiter plate shaker. Return plates to the 20°C incubator 1b. Mark your plate with the correct color 2. Adding the drugs can occasionally kill a few animals per plate, especially if using a solvent other than water. Use an inverted microscope to check for dead animals. Generally, there should be less than 10 dead animals per 96 well plate. Return plates to the 20°C incubator. Day 4: ''' '''Monday (week 3): Change sealers 1. To allow fresh oxygen to enter the culture remove the sealer, wait 1minute and reseal the plate. Shake the plate for 2-3 minutes on a microtiter plate shaker. Repeat once every week. Day 5: Tuesday (week 3): Add new OP50 to prevent starvation 1. Add 5 μL of the 100 mg/ mL OP50 solution that was prepared in part 1 to each of the 96 wells to prevent starvation. Scoring of Lifespans 1. In order to increase the chance that the animals move, shake the 96 well plate on a microtiter plate shaker for 2 min. before counting. 2. In each counting session, record date and number of animals which move as live animals. Movement in liquid is much easier than on solid media and can be induced by strong lights. The use of a higher magnification may help to spot very subtle movements like those of the tip of the pharynx. Such subtle movements are often the only movement observed in very old animals. 3. Return plates to the 20°C incubator 4. Repeat step 2-4 every two to three days until all the animals are dead. MATERIALS: AMPICILLIN P/N: (Sigma-Aldrich, A0166-25G, dessicator in 4°C, #3) AMPHOTERICIN B P/N: (Sigma-Aldrich, A2942, dessicator in -20°C, #2) CARBENICILLIN P/N: (plantMedia, 40310000-3, dessicator in 4°C, #3) FUDR P/N: (Bioworld, 40690016-1, dessicator in 4°C, #3)